The present invention relates to a protein binding rabconnectin-3 and a GDP/GTP exchange protein, and a polynucleotide encoding the same.
Rab3A is a member of a Rab3 family consisting of four members: Rab3A, Rab3B, Rab3C, and Rab3D, and Rab3A is known to play a key regulatory role in Ca2+-dependent exocytosis of neurotransmitters. The process of the Ca2+-dependent exocytosis of neurotransmitters includes the following steps: (1) translocation of synaptic vesicles from the reserve pool to the active zone of the presynaptic plasma membrane where a Ca2+ channel localizes, (2) docking of the vesicles to the active zone, (3) transition from the docking to the priming of the vesicles in the readily releasable pool, and (4) fusion of the vesicles with the membrane induced by Ca2+ influx.
The analysis of Rab3A gene knockout mouse has revealed two actions of Rab3A: (1) it facilitates the translocation and docking of synaptic vesicles to the presynaptic plasma membrane, and (2) it prevents Ca2+-triggered fusion of the vesicles with the plasma membrane. However, the molecular mechanism of these actions of Rab3A in the Ca2+-dependent exocytosis of neurotransmitters is not known.
The Rab3 family members are regulated by three regulators: a GDP dissociation inhibitor (Rab GDI), a GDP/GTP exchange protein (Rab3 GEP), and a GTPase-activating protein (Rab3 GAP). Rab3 GEP and Rab3 GAP are specific for the Rab3 family members, but Rab GDI is active on all the Rab family members. The cyclical activation and inactivation of Rab3A by the action of these regulators are essential for the action of Rab3A in Ca2+-dependent exocytosis of neurotransmitters. For example, a current model for the mode of action of these regulators is as follows: GDP-Rab3A is kept in the cytosol as a complex with Rab GDI. This complex is recruited to synaptic vesicles where GDP-Rab3A is activated to GTP-Rab3A by the action of Rab3 GEP with the help of another unidentified molecule, such as GDI displacement factor (GDF) for Rab5, Rab7, and Rab9, or Rab recycling factor (RRF) for Ypt1 and Ypt7. Neither GDF nor RRF has been isolated. GTP-Rab3A binds its two downstream effectors: rabphilin-3 and Rim-3 localized on the vesicles and the active zone, respectively. Before or after the fusion step, GTP-Rab3A in a complex with the effectors is inactivated to GDP-Rab3A by the action of Rab3 GAP. GDP-Rab3A is trapped by Rab GDI, resulting in the translocation from the vesicles to the cytosol. Thus, it is presumed that Rab3 GEP and Rab3 GAP are recruited to the vesicles when they function; however, their mechanisms remain unknown.
Recently, a novel protein was isolated from a crude synaptic vesicle (CSV) fraction of rat brain by coimmunoprecipitation with Rab3 GEP or Rab3 GAP, and was named rabconnectin-3 (see The Journal of Biological Chemistry, 2002, Vol. 277, No. 12, PP. 9629–9632). Human rabconnectin-3 consists of 3,036 amino acids and shows a calculated molecular weight of 339, 753. Rabconnectin-3 has 12 WD domains. Rabconnectin-3 is abundantly expressed in the brain where it is associated with the synaptic vesicles. Moreover, it has been found that further two proteins are coimmunoprecipitated with Rab3 GEP from a CSV fraction of rat brain (see The Journal of Biological Chemistry, 2002, Vol. 277, No. 12, PP. 9629–9632).